Our overall goal is to clarify the mechanism whereby high dose beta- carotene supplements caused a greater number of lung cancers to occur in smoking populations. Observational epidemiologic studies have demonstrated that individuals with high dietary beta-carotene intakes by virtue of eating fruits and vegetables may have a lower risk of lung cancer. However, two out of three recent human intervention studies using high dose beta-carotene supplements have reported an increase in the risk of lung cancer among smokers. Whether there is a true hazard associated with high dose beta-carotene should be evaluated by controlled studies in both animal and cell models. As described in our Progress Report, our work over the past three years has provided important insights regarding the mechanisms of metabolism of beta- carotene and the possible biologic function of beta-carotene excentric cleavage products. Our laboratory also has a long standing interest in the ferret as model for human beta-carotene metabolism. In preliminary studies, we have shown that high dose beta-carotene causes a down regulation of RARbeta gene expression and a severe alveolar cell proliferation in the lungs of smoke-exposed ferrets. We hypothesize that the free-radical-rich atmosphere in the lung of cigarette smokers alters beta-carotene metabolism and its oxidative metabolites, which, in turn, interfere with retinoid signal transduction and thus accelerate malignant transformation. In the proposed study, we will determine 1) if there is a dose-dependent relationship between beta-carotene and lung premalignant epithelial lesions in cigarette smoke-exposed ferrets in vivo; 2) if smoking alters normal beta-carotene metabolism and the formation of oxidative products; and 3) if high or low dose beta- carotene or its metabolites can down-regulate or interfere with the expression of RARbeta gene, which has tumor suppressor activity. If similar mistakes in the use of chemopreventive agents are to be avoided in the future, we must gain a mechanistic understanding of what went wrong in the beta-carotene experience.